生物支架材料——组织工程连载之二

具有三维结构的支架材料是组织工程的核心内容之一。现有组织工程支架可分为天然生物材料、合成有机材料和无机材料三类。支架材料近年来研究十分活跃,不仅在组织工程的最早产品人工皮肤领域进行了更为完善的研究和开发,同时在诸如人工骨、软骨、神经、血管、皮肤、肝、脾、肾、膀胱等方面进行了大量研究和探索。与普通组织工程支架需要预先制备并在体外成型不同,近年来在骨和软骨组织工程实践中兴起的可注射支架具有许多优势,是未来组织工程支架发展的重要方向之一。     

不同方法制备大鼠肝脏脱细胞支架及其免疫原性研究

目的:肝脏脱细胞支架(decellularized liver bioscaffold,DLB)在组织工程研究中具有良好前景,但对DLB的免疫原性尚未见探讨.本研究利用不同方案制备大鼠DLB,检测支架成分和体内重塑反应,为制备出低免疫原性的DLB提供改进依据.方法:选取文献报道的三种方案(分别以SDS,Triton X-100和NP-40为主要洗脱成分),制备F344大鼠DLB,进行HE和Masson染色,检测DLB中DNA、氨基葡聚糖(GAG)以及羟脯氨酸(HYP)含量;再将DLB埋植于C57BL/6小鼠的背部皮下,于术后第3、7和14天取出后进行形态学观察和组织学评分.结果:三种方案均成功制备出符合脱细胞标准的大鼠DLB;形态学结果提示TritonX-100和NP-40方案较SDS方案能更好地保护肝脏超微结构;成分检测结果发现NP-40方案去除DNA的能力显著强于SDS和Triton X-100方案(P＜0.05),而对GAG含量的洗脱作用最弱(P＜0.05);异种体内埋置实验提示NP-40方案制备的DLB引起的免疫反应强度明显弱于SDS和Triton X-100方案,体内重塑结局评分显著高于SDS和Triton X-100方案.结论:与SDS和TritonX-100方案相比,NP-40方案能更有效地去除肝脏DNA,较好地保留GAG,诱导移植受体对DLB的良性重塑反应,为进一步的体内研究提供更优化的支架.     

应用于组织工程支架制备的电纺技术

组织工程技术为修复病损的组织和器官提供了一种新的途径,在组织工程中,细胞支架起着支撑细胞生长、引导组织再生、控制组织结构和释放活性因子等作用。针对电纺技术的新发展和细胞支架的新理念,综述了国内外利用电纺技术制备细胞支架的工艺条件、制备方法、组织细胞培养等方面的研究进展,并结合作者所在研究团队的研究工作提出了对未来电纺技术在组织工程中应用的研究重点和发展方向的认识。     

软骨基质来源定向结构支架复合软骨细胞体外构建组织工程软骨的研究

目的：探讨采用软骨细胞外基质材料制备的定向结构软骨支架复合软骨细胞,在体外静态培养条件下生成组织工程软骨的可能性。方法：制备牛关节软骨细胞外基质材料,利用温度梯度热诱导相分离技术构建具备垂直定向孔道结构的软骨支架,同时采用传统冷冻干燥方法制备非定向支架,检测两组支架的力学性能;提取兔关节软骨细胞,分别接种两组支架,体外静态培养2周及4周后取材,对构建的组织工程软骨进行组织切片染色、生物化学分析及生物力学检测。结果：定向软骨支架的压缩弹性模量数值明显高于非定向软骨支架,体外培养时定向支架上种子细胞在3-9d内增殖高于非定向支架,差异有统计学意义（P〈0．05）;体外静态培养4周后形成的两组新生组织工程软骨进行软骨特异性染色均呈阳性,在定向组新生软骨切片中在垂直方向上可见大量呈规则平行排列的粗大胶原纤维,两组新生软骨的生物化学检测包括总DNA、总GAG及总胶原含量差异无统计学意义（P〉0．05）。定向组织工程软骨压缩弹性模量在2周及4周时均高于非定向组织工程软骨,差异有统计学意义（P〈0．05）。但两组组织工程软骨上述指标均显著低于正常关节软骨（P〈0．05）。结论：软骨细胞外基质材料制备的定向结构软骨支架复合软骨细胞,在体外静态培养条件下能够成功生成具有定向纤维结构的组织工程软骨,并可以有效促进新生软骨组织力学性能的提升,在软骨组织工程中具有良好的应用前景。     

肌腱组织工程支架与种子细胞研究进展

目的:综述肌腱组织工程支架材料、细胞来源、制备技术及体外构建的研究进展.方法:查阅近期肌腱组织工程研究的相关文献,对组织工程肌腱支架的材料来源、制备技术,复合细胞种类,体外构建力学刺激等进行分析、归纳.结果:肌腱组织工程支架材料有天然材料、人工合成材料及复合材料等;制备技术包括静电纺丝和编织法等;其中支架材料的表面修饰是组织工程化肌腱构建的重要环节.与肌腱材料进行复合的种子细胞有肌腱细胞、骨髓间充质干细胞及成纤维细胞等.结论:复合材料是近年肌腱组织工程支架材料研究的重点,静电纺丝技术是一种具有潜力的支架制备技术,支架材料的表面修饰可促进细胞在支架上的黏附及肌腱的形成,种子细胞的研究仍是肌腱组织工程发展的瓶颈,周期性张力的存在为组织工程化肌腱的形成创造了条件.     

微环境对细胞的影响以及仿生学在组织工程支架中的应用

微环境影响着细胞的增殖、迁移、分化以及细胞功能,细胞微环境影响细胞命运的因素包括细胞之间相互作用、细胞与细胞外基质相互作用、可溶性信号分子以及缺氧和营养对细胞的影响。组织工程支架的制备就是要利用仿生学原理最大程度模拟细胞微环境,从而应用于细胞行为研究以及临床治疗。全面了解细胞微环境对细胞的影响因素是制备组织工程支架的重要条件,而组织工程支架的研究也进一步推动了细胞微环境对细胞影响的认识。组织工程支架研究在组织工程研究中仍具有广阔前景,新的制备工艺也在组织工程支架研究中发挥着巨大推动作用。     

Ⅱ型胶原-硫酸软骨素支架的制备及组织工程软骨的构建

研究经乙基-(3-二甲基氨基丙基)碳化二亚胺盐酸盐(EDC)处理的Ⅱ型胶原-硫酸软骨素支架材料的性能特点,并在体外构建组织工程软骨。从鸡软骨中提取Ⅱ型胶原,以不同浓度的EDC为交联剂通过冷冻干燥的方法制备Ⅱ型胶原与硫酸软骨素复合支架并测定其理化性质。将体外培养的新生兔关节软骨细胞接种在Ⅱ型胶原与硫酸软骨素复合支架上,观察软骨细胞在支架上的生长形态并检测支架上软骨细胞分泌的糖胺聚糖含量及Ⅱ型胶原含量。结果表明:采用EDC与硫酸软骨素交联增加了支架的稳定性,最适的交联剂质量浓度为7 mg/mL。软骨细胞在复合支架上增殖分化良好,并保持软骨细胞特异分化的表型,分泌Ⅱ型胶原与蛋白多糖(GAG)。培养14 d后已有软骨样组织形成。     

冻融联合灌注法优化大鼠肾脏脱细胞支架的制备

本研究旨在探索优化肾脏脱细胞支架的制备方法,为肾脏组织工程及肾脏体外病理、毒理研究提供实验基础。取大鼠肾脏灌注PBS作为对照组 (Control组),在不同流速下分别以十二烷基磺酸钠 (Sodium dodecyl sulfate,SDS) 灌注 (S组),Triton X-100联合SDS灌注 (TS组),反复冻融后Triton X-100联合SDS灌注(FTS组),制备肾脏脱细胞支架,并测定其流体分布及脉管阻力。HE染色、DAPI染色、DNA定量检测脱细胞支架脱细胞程度,Masson染色、PAS染色、免疫组织化学染色检测脱细胞支架主要成分的保留和结构的完整,扫描电镜检测支架的超微结构,MTT法检测支架的细胞毒性,ELISA检测支架中生长因子的含量。结果显示,FTS组脱细胞用时较S组、TS组少,10 mL/min组支架脉管阻力较低,S组、TS组、FTS组流体分布与Control组存在差异。HE染色和DAPI染色显示各组支架未见细胞成分残留,DNA含量＜50 ng/mg。Masson染色和PAS染色可见细胞外网状胶原及多糖,免疫组织化学染色见Ⅰ型胶原 (CollagenⅠ)、Ⅳ型胶原蛋白 (Collagen Ⅳ)、纤维连接蛋白 (Fibronectin)、层粘连蛋白 (Laminin) 表达。扫描电镜见支架呈蜂窝状结构。MTT法检测支架细胞毒性分级在0–1级之间。ELISA检测提示FTS组VEGF、EGF、IGF-1、PDGF含量明显高于S组和TS组。综上,联合冻融和灌注法能够制备更为理想且有效的大鼠肾脏整器官脱细胞支架,为肾脏组织工程及肾脏体外病理、毒理学研究奠定基础。     

组织工程心脏瓣膜的研究进展

组织工程心脏瓣膜(tissue engineering heart valve,TEHV)理论上能克服机械瓣及生物瓣的不足,具有广阔的发展前景。目前组织工程心脏瓣膜的研究主要集中在瓣膜支架材料的选取及制备、种子细胞的选择和种子细胞的种植及培养等三方面。本文将分别就这三方面研究进展进行介绍,分析目前存在的问题,并对其应用进行展望。     

制备大孔径静电纺丝组织工程支架的研究进展

研究表明静电纺丝可以制备出模拟细胞外基质的三维结构,其中限制静电纺丝纤维支架应用的问题之一就是纤维排列紧密导致支架的孔径较小,从而阻碍了细胞的浸入,组织中血管化的形成以及支架与宿主细胞的融合。为了增大支架的孔径,提高孔隙率,许多研究者提出了相应的策略。本文综述了多种制备大孔径静电纺丝纤维支架的方法,主要包括不同接收装置控制电场分布、盐粒子/聚合物析出法、水浴接收、低温静电纺丝以及激光/紫外烧蚀法等,以上的方法都能够有效的增大静电纺丝三维支架的孔径,进而提高了细胞的浸润性、营养物质的传输以及废物的排出,为静电纺丝纤维支架在组织工程中的应用奠定了基础。     

Hyaluronan is an abundant constituent of the extracellular matrix of Xenopus embryos

The spatiotemporal distribution of hyaluronan (HA), a major constituent of the vertebrate extracellular matrix, was analyzed during early embryonic development of Xenopus laevis. This polysaccharide is abundantly present in ventricular structures such as the blastocoel, the archenteron as well as later on in the hepatic cavity, the brain ventricles and the developing heart. At the blastula stage, HA was detected in the extracellular matrix of the ecto- and mesodermal primordia. Shortly before gastrulation, it becomes enriched at the basal site of the superficial cell layer of the ectoderm. During gastrulation, enhanced synthesis of HA takes place in the involuting marginal zone, shortly before invagination starts, hence, resulting in a torus-like deposition in the deep layer of the equatorial mesodermal primordium. After gastrulation, HA appears to accumulate within the extracellular matrix demarcating the primary germ layers. During tailbud stages, it is found highly enriched in many mesodermal derivatives, e.g., in mesenchyme, the heart, precordal cartilage and the lung primordia. Furthermore, extracellular matrix of the ventral mesodermal cell layer in the trunk region and the immediate proximity of blood vessels contain high amounts of HA.     

Restoration of senescent human diploid fibroblasts by modulation of the extracellular matrix

Human diploid fibroblasts have the capacity to complete a finite number of cell divisions before entering a state of replicative senescence characterized by growth arrest, changes in morphology, and altered gene expression. Herein, we report that interaction with extracellular matrix (ECM) from young cells is sufficient to restore aged, senescent cells to an apparently youthful state. The identity of the restored cells as having been derived from senescent cells has been confirmed by a variety of methods, including time lapse live cell imaging and DNA finger print analysis. In addition to cell morphology, phenotypic restoration was assessed by resumption of proliferative potential, growth factor responsiveness, reduction of intracellular reactive oxygen species levels, recovery of mitochondrial membrane potential, and increased telomere length. Mechanistically, we find that both Ku and SIRT1 are induced during restoration and are required for senescent cells to return to a youthful phenotype. These observations demonstrate that human cellular senescence is profoundly influenced by cues from the ECM, and that senescent cell plasticity is much greater than that was previously believed to be the case.     

Accumulation of loading damage and unloading reperfusion injury — Modeling of the propagation of deep tissue ulcers

Deep tissue injury (DTI) occurs in deep muscles around bony prominences due to excessive and prolonged mechanical loading acting on the skin surface. The condition is clinically challenging because it can escape being noticed till the damage propagates all the way to the skin. In this study, a semi-3D finite element model of a human buttock was used to simulate the process of ulcer evolution based on our recent damage accumulation and repair theory for DTI. The theory included not only the loading damage, but also further reperfusion and inflammatory injuries upon unloading. The results showed that depending on the model parameters and loading conditions, a deep tissue ulcer may initiate around a bony prominence and expand to affect the entire tissue thickness. The damage evolution can be affected by the tissue healing rate, the loading–unloading pattern and the cushion stiffness. The results may help clinical workers appreciate the importance of proper patient turning and the appropriate choice of cushion.     

Morphofunctional characterization of decellularized vena cava as tissue engineering scaffolds

Clinical experience for peripheral arterial disease treatment shows poor results when synthetic grafts are used to approach infrapopliteal arterial segments. However, tissue engineering may be an option to yield surrogate biocompatible neovessels. Thus, biological decellularized scaffolds could provide natural tissue architecture to use in tissue engineering, when the absence of ideal autologous veins reduces surgical options. The goal of this study was to evaluate different chemical induced decellularization protocols of the inferior vena cava of rabbits. They were decellularized with Triton X100 (TX100), sodium dodecyl sulfate (SDS) or sodium deoxycholate (DS). Afterwards, we assessed the remaining extracellular matrix (ECM) integrity, residual toxicity and the biomechanical resistance of the scaffolds. Our results showed that TX100 was not effective to remove the cells, while protocols using SDS 1% for 2 h and DS 2% for 1 h, efficiently removed the cells and were better characterized. These scaffolds preserved the original organization of ECM. In addition, the residual toxicity assessment did not reveal statistically significant changes while decellularized scaffolds retained the equivalent biomechanical properties when compared with the control. Our results concluded that protocols using SDS and DS were effective at obtaining decellularized scaffolds, which may be useful for blood vessel tissue engineering.     

Biophysical factors in the regulation of asymmetric division of stem cells

Stem cells are a promising cell source for regenerative medicine due to their characteristics of self‐renewal and differentiation. The intricate balance between these two cell fates is maintained by precisely controlled symmetric and asymmetric cell divisions. Asymmetric division has a fundamental importance in maintaining tissue homeostasis and in the development of multi‐cellular organisms. For example, during development, asymmetric cell divisions are responsible for the formation of the body axis. Mechanistically, mitotic spindle dynamics determine the assembly and separation of chromosomes and regulate the orientation of cell division. Interestingly, symmetric and asymmetric cell division is not mutually exclusive and a range of factors are involved in such cell‐fate decisions, the measurement of which can provide efficient and reliable information on the regenerative potential of a cell. The balance between self‐renewal and differentiation in stem cells is controlled by various biophysical and biochemical cues. Although the role of biochemical factors in asymmetric stem cell division has been widely studied, the effect of biophysical cues in stem‐cell self‐renewal is not comprehensively understood. Herein, we review the biological relevance of stem‐cell asymmetric division to regenerative medicine and discuss the influences of various intrinsic and extrinsic biophysical cues in stem‐cell self‐renewal. This review particularly aims to inform the clinical translation of efforts to control the self‐renewal ability of stem cells through the tuning of various biophysical cues.     

Matrix metalloproteinases regulate mesonephric cell migration in developing XY gonads which correlates with the inhibition of tissue inhibitor of metalloproteinase-3 by Sry

In the mouse, the sex determining gene Sry, on the Y chromosome, controls testis differentiation during embryogenesis. Following Sry expression, indifferent XY gonads increase their size relative to XX gonads and form cord-like structures with the adjacent mesonephros, providing XY gonad somatic cells. This mesonephric cell migration is known to depend on Sry, but the molecular mechanism of mesonephric cell migration remains unknown. In this study, it was shown that cells expressing Sry induced proliferation of mesonephric cells migrating into male gonads, and inhibited expression of the tissue inhibitor of metalloproteinases (TIMP)-3 gene, which is the endogenous inhibitor of matrix metalloproteinases (MMP). In addition, the mesonephric cell migration was blocked by a chemically synthesized inhibitor of MMP in a gonad/mesonephros organ co-culture system with enhanced green fluorescent protein transgenic embryos. The findings indicate that MMP may play a critical role in mesonephric cell migration, and the function of MMP may be regulated by a Sry-TIMP-3 cascade. These findings are an important clue for the elucidation of testicular formation in developing gonads.     

Engineering of human hepatic tissue with functional vascular networks

Although absolute organ shortage highlights the needs of alternative organ sources for regenerative medicine, the generation of a three-dimensional (3D) and complex vital organ, such as well-vascularized liver, remains a challenge. To this end, tissue engineering holds great promise; however, this approach is significantly limited by the failure of early vascularization in vivo after implantation. Here, we established a stable 3D in vitro pre-vascularization platform to generate human hepatic tissue after implantation in vivo. Human fetal liver cells (hFLCs) were mixed with human umbilical vein endothelial cells (HUVECs) and mesenchymal stem cells (hMSCs) and were implanted into a collagen/fibronectin matrix composite that was used as a 3-D carrier. After a couple of days, the fluorescent HUVECs developed premature vascular networks in vitro, which were stabilized by hMSCs. The establishment of functional vessels inside the pre-vascularized constructs was proven using dextran infusion studies after implantation under a transparency cranial window. Furthermore, dynamic morphological changes during embryonic liver cell maturation were intravitaly quantified with high-resolution confocal microscope analysis. The engineered human hepatic tissue demonstrated multiple liver-specific features, both structural and functional. Our new techniques discussed here can be implemented in future clinical uses and industrial uses, such as drug testing.     

Engineering of human hepatic tissue with functional vascular networks

Although absolute organ shortage highlights the needs of alternative organ sources for regenerative medicine, the generation of a three-dimensional (3D) and complex vital organ, such as well-vascularized liver, remains a challenge. To this end, tissue engineering holds great promise; however, this approach is significantly limited by the failure of early vascularization in vivo after implantation. Here, we established a stable 3D in vitro pre-vascularization platform to generate human hepatic tissue after implantation in vivo. Human fetal liver cells (hFLCs) were mixed with human umbilical vein endothelial cells (HUVECs) and mesenchymal stem cells (hMSCs) and were implanted into a collagen/fibronectin matrix composite that was used as a 3-D carrier. After a couple of days, the fluorescent HUVECs developed premature vascular networks in vitro, which were stabilized by hMSCs. The establishment of functional vessels inside the pre-vascularized constructs was proven using dextran infusion studies after implantation under a transparency cranial window. Furthermore, dynamic morphological changes during embryonic liver cell maturation were intravitaly quantified with high-resolution confocal microscope analysis. The engineered human hepatic tissue demonstrated multiple liver-specific features, both structural and functional. Our new techniques discussed here can be implemented in future clinical uses and industrial uses, such as drug testing.     

The significance of alpha 5 beta 1 integrin dependent and independent actin cytoskelton organization in cell transformation and survival

Cell-matrix and cell-cell interactions are important physiological determinants of cell growth, survival and transformation. Cell adhesion to the extra cellular matrix (ECM) via integrins also crucially influences the organization of the cytoskeleton. It triggers a cascade of intracellular biochemical events, which regulate cell viability and growth. We have studied the relationship between cell attachment to the substratum and cytoskeletal organization and cell survival and transformation. Our results demonstrate that in the absence of attachment to the substratum, adhesion-dependent fibroblasts exhibit rapid loss of viability. However, a small percentage of cells survive even after remaining non-adherent for 16h. The adherent and non-adherent cells differ from one another both morphologically and physiologically. The latter show a loss of alpha5beta1 integrin expression on their surface and bind non-specifically to the substratum and ECM, thereby activating certain pathways more efficiently than adherent cells. We have also shown that non-adherent cells grow faster and have worse cytoskeletal organization after attachment to the substratum, and do not form focal adhesions or actin stress fibres. Hence, our data suggests that rat fibroblasts in prolonged suspension exhibit some properties that are comparable to cells undergoing transformation, by adapting integrin-dependent or independent signalling pathways for their survival.